Overview of noncoding RNAs involved in the osteogenic differentiation of periodontal ligament stem cells.

Periodontal diseases are infectious diseases that are characterized by progressive damage to dental support tissue. The major goal of periodontal therapy is to regenerate the periodontium destroyed by periodontal diseases. Human periodontal ligament (PDL) tissue possesses periodontal regenerative properties, and periodontal ligament stem cells (PDLSCs) with the capacity for osteogenic differentiation show strong potential in clinical application for periodontium repair and regeneration. Noncoding RNAs (ncRNAs), which include a substantial portion of poly-A tail mature RNAs, are considered "transcriptional noise." Recent studies show that ncRNAs play a major role in PDLSC differentiation; therefore, exploring how ncRNAs participate in the osteogenic differentiation of PDLSCs may help to elucidate the underlying mechanism of the osteogenic differentiation of PDLSCs and further shed light on the potential of stem cell transplantation for periodontium regeneration. In this review paper, we discuss the history of PDLSC research and highlight the regulatory mechanism of ncRNAs in the osteogenic differentiation of PDLSCs.

Plantamajoside attenuates inflammatory response in LPS-stimulated human gingival fibroblasts by inhibiting PI3K/AKT signaling pathway.

Periodontitis is an important inflammatory disease that often causes by periodontopathic bacteria. The present study, we tested the anti-inflammatory effects of plantamajoside on LPS-stimulated human gingival fibroblasts. Human gingival fibroblasts (HGFs) were stimulated with LPS from Porphyromonas gingivalis. Plantamajoside was administrated 1 h before LPS treatment. The results demonstrated that plantamajoside decreased the production of PGE2, NO, IL-6, and IL-8 in LPS-stimulated HGFs. LPS-induced NF-κB p65 and IκB phosphorylation were also suppressed by plantamajoside. Furthermore, plantamajoside inhibited LPS-induced PI3K and AKT phosphorylation. In conclusion, these results suggested that the mechanism of plantamajoside was through inhibiting PI3K/AKT signaling pathway, which lead to the inhibition of NF-κB activation and inflammatory response.

A novel small deletion mutation in RUNX2 gene in one Chinese family with cleidocranial dysplasia.

Cleidocranial dysplasia (CCD) is a skeletal dysplasia with autosomal-dominant inheritance. The runt related transcription factor 2 (RUNX2) gene is the only gene in which mutations are known to cause CCD. We report identification of a novel small deletions mutation in the RUNX2 gene in a Chinese family with CCD. A 29-year-old female was diagnosed as proband of CCD based on the clinical findings, which show delayed closure of the fontanels, hypoplastic or aplastic clavicles and dental anomalies. Similar dental and skeletal symptoms were also observed in the other three affected individuals. We prepared genomic DNA from all four affected individuals, unaffected individual from her family members, as well as 100 unrelated healthy controls. PCR was conducted using the above genomic DNA as template and the RUNX2 gene-specific primers. The PCR product was subjected to direct sequencing and the sequence was compared to that of RUNX2 gene within the NCBI database. We detected a small deletion CCTA from nucleotide 635 to nucleotide 638 in exon 3 of RUNX2 gene of the proband. This will lead to the introduction of a translational stop codon at codon 220, resulting in a truncated RUNX2 protein, and therefore within the runt domain of the RUNX2 protein. We detected the same mutation in the the other three affected individuals, and did not detect any mutation in the unaffected family members or the 100 unrelated healthy controls, demonstrating that this is a novel missense mutation in RUNX2 gene and therefore, contributes to the molecular diagnosis of CCD.

RCCS enhances EOE cell proliferation and their differentiation into ameloblasts.

In this article we report on the culturing of dental enamel organ epithelia (EOE) using a rotary cell culture system (RCCS) bioreactor associated with a cytodex-3 microcarrier. This culture system enhanced the proliferation and differentiation of the EOE into ameloblasts. Primary dental EOE trypsinized from 4-day old post-natal rat pups were cultured in the RCCS associated with Cytodex-3. The results were analyzed in comparison to a conventional plate system (control). Cells grown in RCCS have shown higher viabilities (above 90%) and final cell densities in terms of cells/ml than in the control system. In the case of RCCS, 46±2 manifold increases were obtained, while significantly lower yields of 10.8±2.5 manifod were obtained for control plates. Throughout the experiments, glucose levels were maintained within the accepted physiological range. In this case, LDH levels are kept low (below 150 mmol/ml), which is in accordance with the low cell death observed in the RCCS. Scanning electron microscopy revealed cells that were spread and forming three dimensional aggregates on the surface of cytodex-3. Cells cultured in the RCCS exhibited a stronger positive immunofluorescence staining for ameloblastin than those in control plates. RT-PCR results revealed that cells cultured in RCCS have higher amelogenin mRNA levels compared to controls. We have done an exploratory study on biological characteristics and self-assembling of epithelium cellula intersitialis, which demonstrated that the special 3D environment enhanced the rat dental EOE cell proliferation and differentiation into ameloblasts. The study has revealed that RCCS could be used to study the reaction of the EOE cells, tooth enamel organ cells and mesenchymal cells under the spacial 3D culture system, which will also provide a novel hypothesis for dental regeneration.

Disturbed tooth germ development in the absence of MINT in the cultured mouse mandibular explants.

The Msx2-interacting nuclear target protein (MINT) is a nuclear matrix protein that regulates the development of many tissues. However, little is known regarding the role of MINT in tooth development. In this study, we prepared polyclonal antibodies against MINT, and found that that MINT was expressed in different cells at each stage of tooth germ development by immunohistochemistry. The role of MINT in tooth development was further illustrated by the misshapen and severely hypoplastic tooth organ in the cultured mandibular explants of MINT deficient mice. From the initiation to cap stage, the differences between mutants and wild-type molars were more and more distinguished histologically. In the MINT-deficient mandibular explants, the tooth germ was reduced in the overall size and lacked enamel knot, with abnormal dental lamina and collapsed stellate reticulum. Furthermore, the BrdU incorporation experiment showed that the proliferation activity was significantly reduced in MINT-deficient dental epithelium. Our results suggest that MINT plays an important role in tooth development, in particular, epithelial morphogenesis.

Inhibition of RANK/RANKL signal transduction pathway: a promising approach for osteoporosis treatment.

Osteoporosis is a bone disease causing impaired bone strength. It is characterized by increased osteoclast formation or enhanced bone resorption, leading to an increased risk of fragility fractures. Its prevalence increases with age. The advent of an aging population suggests that progressively more individuals will develop this disease in the aging population. A number of drugs for the prevention and treatment of osteoporosis act by inhibiting bone resorption. However, the effectiveness of osteoporosis treatment in clinical practice is limited. Since the osteoclast is the only cell in the body that is capable of resorbing bone, understanding its biology will be necessary for developing a new therapeutic approach for osteoporosis. Recently, it was discovered that the receptor activator of nuclear factor kappaB (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) system is an important signal transduction pathway that regulates osteoclast formation. The binding of OPG to RANKL inhibits the binding between RANKL and RANK; this, in turn, prevents osteoclast precursors from differentiating and fusing to form mature osteoclasts. Therefore, the inhibition of the RANK/RANKL pathway inhibits osteoclast formation, differentiation, activation, and bone resorption. A potential clinical antiresorptive therapy can be developed by using an anti-RANKL monoclonal antibody, such as denosumab, that binds to RANKL with high affinity and specificity and blocks RANKL-RANK interactions.

[Cbfa1 induces the expression of the mineral-related proteins in human dental papilla cells].

OBJECTIVE: To explicit whether the expression of the mineral-related proteins is regulated by cbfa1 in human dental papilla cells. METHODS: Human dental papilla cells were cultured in vitro and transfected with pcDNA3-cbfa1 recombinant plasmids. After selected with G418 sulfate, a cell clone named PC-3, which could stably express the cbfa1 mRNA and protein, was proved by PCR and western blot. Then the amount of ALP and OC and the expression of OPN, BSP, ON, DMP1, DSP and DSPP were detected by immunohistochemistry, Western blot and PCR methods. RESULTS: We established the human dental papilla cells model PC-3 which could stably express the cbfa1 mRNA and protein. Compared with normal cells, a lot of mineral-related proteins such as ALP, OC, OPN, BSP, ON, DMP1 were upregulated in PC-3 cells. CONCLUSIONS: In human dental papilla cells, cbfa1 can induce the expression of most mineral-related genes and proteins. It may implicate that cbfa1 must play a key role during tooth development and mineralization.